Ambrogelly Alexandre, Frugier Magali, Ibba Michael, Söll Dieter, Giegé Richard
Department of Molecular Biophysics and Biochemistry, New Haven, CT 06520-8114, USA.
FEBS Lett. 2005 May 9;579(12):2629-34. doi: 10.1016/j.febslet.2005.04.001. Epub 2005 Apr 9.
Borrelia burgdorferi and other spirochetes contain a class I lysyl-tRNA synthetase (LysRS), in contrast to most eubacteria that have a canonical class II LysRS. We analyzed tRNA(Lys) recognition by B. burgdorferi LysRS, using two complementary approaches. First, the nucleotides of B. burgdorferi tRNA(Lys) in contact with B. burgdorferi LysRS were determined by enzymatic footprinting experiments. Second, the kinetic parameters for a series of variants of the B. burgdorferi tRNA(Lys) were then determined during aminoacylation by B. burgdorferi LysRS. The identity elements were found to be mostly located in the anticodon and in the acceptor stem. Transplantation of the identified identity elements into the Escherichia coli tRNA(Asp) scaffold endowed lysylation activity on the resulting chimera, indicating that a functional B. burgdorferi lysine tRNA identity set had been determined.
与大多数拥有典型II类赖氨酰 - tRNA合成酶(LysRS)的真细菌不同,伯氏疏螺旋体和其他螺旋体含有I类赖氨酰 - tRNA合成酶(LysRS)。我们使用两种互补方法分析了伯氏疏螺旋体LysRS对tRNA(Lys)的识别。首先,通过酶足迹实验确定了与伯氏疏螺旋体LysRS接触的伯氏疏螺旋体tRNA(Lys)的核苷酸。其次,在伯氏疏螺旋体LysRS进行氨酰化反应期间,测定了一系列伯氏疏螺旋体tRNA(Lys)变体的动力学参数。发现识别元件大多位于反密码子和接受茎中。将鉴定出的识别元件移植到大肠杆菌tRNA(Asp)支架中,赋予了所得嵌合体赖氨酰化活性,表明已确定了功能性的伯氏疏螺旋体赖氨酸tRNA识别集。